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Functional and Structural Characterization of alpha-(1 -> 2) Branching Sucrase Derived fromDSR-E GlucansucraseBrison, Yoann; Pijning, Tjaard; Malbert, Yannick; Fabre, Emeline; Mourey, Lionel; Morel,Sandrine; Potocki-Veronese, Gabrielle; Monsan, Pierre; Tranier, Samuel; Remaud-Simeon,MagaliPublished in:The Journal of Biological Chemistry

DOI:10.1074/jbc.M111.305078

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Citation for published version (APA):Brison, Y., Pijning, T., Malbert, Y., Fabre, E., Mourey, L., Morel, S., ... Dijkstra, B. W. (2012). Functional andStructural Characterization of alpha-(1 -> 2) Branching Sucrase Derived from DSR-E Glucansucrase. TheJournal of Biological Chemistry, 287(11), 7915-7924. DOI: 10.1074/jbc.M111.305078

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Functional and Structural Characterization of �-(132)Branching Sucrase Derived from DSR-E Glucansucrase*□S

Received for publication, September 16, 2011, and in revised form, January 5, 2012 Published, JBC Papers in Press, January 18, 2012, DOI 10.1074/jbc.M111.305078

Yoann Brison‡§¶, Tjaard Pijning�, Yannick Malbert‡§¶, Émeline Fabre‡§¶1, Lionel Mourey**‡‡, Sandrine Morel‡§¶,Gabrielle Potocki-Véronèse‡§¶, Pierre Monsan‡§¶§§, Samuel Tranier**‡‡2, Magali Remaud-Siméon‡§¶3,and Bauke W. Dijkstra�4

From the ‡Université de Toulouse, INSA, UPS, INP, LISBP, F-31077 Toulouse, France, §CNRS UMR 5504, F-31400 Toulouse, France,¶INRA UMR 792 Ingénierie des Systèmes Biologiques et des Procédés, F-31400 Toulouse, France, the �Laboratory of BiophysicalChemistry, University of Groningen, Nijenborgh 7, 9747 AG Groningen, The Netherlands, the **Institut de Pharmacologie et deBiologie Structurale, Centre National de la Recherche Scientifique, 31077 Toulouse, France, the ‡‡Université de Toulouse, UniversitéPaul Sabatier, Institut de Pharmacologie et de Biologie Structurale, 31077 Toulouse, France, and the §§Institut Universitaire deFrance, 75005 Paris, France

Background:The transglucosidaseGBD-CD2 shows a unique�-(132) branching specificity amongGH70 familymemberswhen catalyzing dextran glucosylation from sucrose.Results: The truncated form �N123-GBD-CD2 was biochemically studied and structurally characterized at 1.90 Å resolution.Conclusion: Dextran recognition and regiospecificity clearly involves a residue in subsite �1.Significance: This is the first three-dimensional structure of a GH70 enzyme that reveals determinants of �-(132) linkagespecificity.

�N123-glucan-binding domain-catalytic domain 2 (�N123-GBD-CD2) is a truncated formof thebifunctional glucansucraseDSR-E from Leuconostoc mesenteroides NRRL B-1299. It wasconstructed by rational truncation of GBD-CD2, which harborsthe second catalytic domain of DSR-E. Like GBD-CD2, this var-iant displays �-(132) branching activity when incubated withsucrose as glucosyl donor and (oligo-)dextran as acceptor, trans-ferring glucosyl residues to the acceptor via a ping-pong bi-bimechanism. This allows the formation of prebiotic moleculescontaining controlled amounts of�-(132) linkages. The crystalstructure of the apo �-(132) branching sucrase �N123-GBD-CD2 was solved at 1.90 Å resolution. The protein adopts theunusual U-shape fold organized in five distinct domains, alsofound in GTF180-�N and GTF-SI glucansucrases of glycosidehydrolase family 70. Residues forming subsite �1, involved inbinding the glucosyl residue of sucrose and catalysis, are strictlyconserved in both GTF180-�N and �N123-GBD-CD2. Subsite�1 analysis revealed three residues (Ala-2249, Gly-2250, and

Phe-2214) that are specific to �N123-GBD-CD2. Mutation ofthese residues to the corresponding residues found inGTF180-�N showed that Ala-2249 and Gly-2250 are notdirectly involved in substrate binding and regiospecificity. Incontrast, mutant F2214N had lost its ability to branch dextran,although it was still active on sucrose alone. Furthermore, threeloops belonging to domains A and B at the upper part of thecatalytic gorge are also specific to �N123-GBD-CD2. These dis-tinguishing features are also proposed to be involved in the cor-rect positioning of dextran acceptormolecules allowing the for-mation of �-(132) branches.

Glucansucrases from glycoside hydrolase family 70 (GH70)5are transglucosidases produced by lactic acid bacteria from thegenera Leuconostoc, Lactobacillus, Streptococcus, Weissella,andOenococcus (1). They naturally catalyze the polymerizationof glucosyl residues with concomitant fructose release fromsucrose, a cheap agroresource. Depending on the enzyme spec-ificity, a large variety of glucans containing all types of gluco-sidic bonds, namely �-(132), �-(133), �-(134), or �-(136),and varying in terms of size, structure, degree of branching, andspatial arrangement are synthesized. These enzymes are alsoable to transfer the glucosyl unit from sucrose onto hydroxy-lated acceptor molecules added in the reaction medium andstand as very attractive biocatalysts for the production of novelbiopolymers, prebiotic oligosaccharides, and new glucoderiva-tives (2).Sequence analysis, functional characterization, and protein

engineering showed that glucansucrases are structurally andmechanistically related to GH family 13 (3). They are �-retain-

* This work was supported by the region Midi-Pyrénées (France) and theEuropean Molecular Biology Organization.

□S This article contains supplemental text, Tables S1–S3, and Figs. S1–S9.The atomic coordinates and structure factors (codes 3TTO and 3TTQ) have been

deposited in the Protein Data Bank, Research Collaboratory for StructuralBioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

1 Present address: Unité de Glycobiologie Structurale et Fonctionnelle, CNRSUMR 8576, IFR 147, Université Lille 1, Sciences et Technologies, 59655 Vil-leneuve d’Ascq cedex, France.

2 To whom correspondence may be addressed: Institut de Pharmacologie etde Biologie Structurale, 205 route de Narbonne, 31077 Toulouse, France.Tel.: 33-561-175-438; Fax: 33-561-175-994; E-mail: Samuel.Tranier@ipbs.fr.

3 To whom correspondence may be addressed: INSA, LISBP, 135 avenue deRangueil, 31077 Toulouse, France. Tel.: 33-561-559-446; Fax: 33-561-559-400; E-mail: remaud@insa-toulouse.fr.

4 To whom correspondence may be addressed: Laboratory of BiophysicalChemistry, University of Groningen, Nijenborgh 7, 9747 AG Groningen,The Netherlands. Tel.: 31-503-634-381; Fax: 31-503-634-800; E-mail: b.w.dijkstra@rug.nl.

5 The abbreviations used are: GH, glycoside hydrolase; GBD, glucan-bindingdomain; GBD-CD2, GBD-catalytic domain 2; Glcp, glucopyranose.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 287, NO. 11, pp. 7915–7924, March 9, 2012© 2012 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.

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http://www.jbc.org/content/suppl/2012/01/18/M111.305078.DC1.html Supplemental Material can be found at:

ing enzymes and sucrose cleavage is predicted to occur throughthe formation of a �-D-glucosyl covalent intermediate. Thisreaction involves one unique active site and requires the con-certed action of an aspartate and a glutamic acid, which act asthe nucleophile and the acid-base catalyst, respectively (4). Sec-ondary structure predictions suggested that the catalyticdomain of GH70 glucansucrases consists of a circularly per-muted (�/�)8 barrel compared with that of GH13 familyenzymes (5). These predictions have been recently confirmedby the elucidation of the three-dimensional structures of theGH70 glucansucrases GTF180-�N and GTF-SI (6–9).GTF180-�N is a dextransucrase synthesizing mainly �-(136)glucosidic linkages, whereas GTF-SI mutansucrase is specificfor �-(133) bond formation.In the GH70 family, the enzyme DSR-E from Leuconostoc

mesenteroidesNRRL B-1299 drew our attention because it wasone of the rare enzymes able to synthesize dextrans with highamounts of �-(132) branch linkages. Sequence analysis of thisvery large enzyme (313 kDa) revealed the presence of two cat-alytic domains, CD1 and CD2, separated by a glucan-bindingdomain (GBD). CD1 and CD2, which share 45% identity and65% similarity, were both classified in family GH70 (10). Bothcontain the highly conserved amino acids proposed to beinvolved in the formation of the glucosyl enzyme intermediate.Biochemical characterization of two recombinant truncatedforms (CD1-GBD and GBD-CD2) showed that CD1-GBD actsas a polymerase, producing a glucan containing 86% �-(136),11% �-(133), and 3% �-(134) glucosidic bonds. The secondform (GBD-CD2) was found to be exclusively responsible forthe synthesis of�-(132) linkages (11). Indeed, this enzyme actsas a very efficient transglucosidase in the presence of sucroseand either linear �-(136) glucans (dextrans) or linear gluco-oligosaccharides, which are used as acceptors. Steady-statekinetic analysis of �-(132) branch formation revealed that theenzyme displays a ping-pong bi-bimechanism (12). In addition,experimental conditions have been established that enable theproduction of new dextrans with controlled sizes and �-(132)linkage contents (12). The presence of �-(132) linkages ren-ders these products resistant to the action ofmammalian diges-tive enzymes and promotes the growth of beneficial bacteria ofthe gut microbiome (13–17).Because of this unique specificity, GBD-CD2 holds a great

potential for the production of novel functional foods. To fur-ther investigate structure-function relationships of this�-(132) branching sucrase, we performed rational truncationsof GBD-CD2 to obtain a pure and crystallizable enzyme form.The specificity and kinetic properties of the variant �N123-GBD-CD2 were investigated, and the apo x-ray structure wassolved at 1.90 Å resolution. Additionally, the x-ray structure ofthis enzyme was solved at 3.3 Å resolution in a different crystalform. These are the first three-dimensional structures of an�-(132) branching sucrase. When compared with theGTF180-�N glucansucrase and GTF-SI mutansucrase struc-tures, the GBD-CD2 structure revealed common but also verydistinctive features that are discussed with regard to the�-(132) branching properties.

EXPERIMENTAL PROCEDURES

Production of �N123-GBD-CD2—The gbd-cd2 gene insertedinto pBADTOPOTAvector (Invitrogen)was amplified by PCRusing the forward primer CACCATGGCACAAGCAGGT-CACTATATCACGAAAA and reverse primer AGCTTGAG-GTAATGTTGATTTATC for �N123-gbd-cd2. The primersused to generate other truncated mutants are listed in supple-mental Table S1. The purified PCR products were cloned into apBAD TOPO Directional 102 vector (Invitrogen). After liga-tion, the N-terminal thioredoxin tag was removed by NcoIrestriction endonuclease digestion (supplemental Table S1).The constructs resulted in proteins with a C-terminal V5epitope His6 tag. The Pfu Turbo polymerase (Stratagene) andall restriction enzymes (New England Biolabs) were usedaccording to themanufacturers’ instructions. DNA sequencingof the �N123-gbd-cd2 gene did not reveal any mutation (Mille-Gen, Labège, France). Transformed Escherichia coli strainTOP10 One shot (Invitrogen) was grown in Luria-Bertanimedium supplemented with 100 �g/ml ampicillin. Inductionwith 0.02% (w/v) L-arabinose was performed at an A600 nm of0.5. The cellswere grown for an 8 additional hours, harvested bycentrifugation, resuspended in PBS buffer, pH 7.0, supple-mented with EDTA free anti-protease tablets (Roche), and dis-rupted by sonication. The�N123-GBD-CD2 enzymewas recov-ered as inclusion bodies in the crude cell extract.Purification of �N123-GBD-CD2—Inclusion bodies were

recovered by three cycles of washing, with PBS buffer supple-mented with 1 mM EDTA and 1% (v/v) Triton X-100, followedby centrifugation at 20,000 � g for 20 min at 4 °C. They weredenaturated using 8 M urea buffer, pH 8.0. The denatured pro-tein preparation (120 ml) was supplemented with 500 mM

NaCl, 0.5% (v/v) glycerol, and 25 mM imidazole, adjusted to pH7.4, and centrifuged at 20,000 � g for 20 min. The supernatantwas injected at a flow rate of 3 ml/min onto a 20-ml nickel-nitrilotriacetic acid-Sepharose column (GEHealthcare Life Sci-ences), equilibratedwith 8M urea buffer, pH 8.0. Protein refold-ing was carried out on-column, with five column volumes ofbuffer (20 mM sodium phosphate, 500 mM NaCl, 1% (v/v) glyc-erol, pH 7.4). The protein was eluted with a gradient of imidaz-ole and dialyzed overnight at 4 °C against 20 mM sodium phos-phate, 25 mMNaCl, and 1% (v/v) glycerol, pH 7.4. The solutionwas then applied to Q-Sepharose resin (GE Healthcare) andeluted using a gradient of 25–500 mM NaCl in 20 mM sodiumphosphate, pH 7.4, supplemented with 1% (v/v) glycerol. Theeluted protein was dialyzed overnight at 4 °C against 20 mM

sodium acetate, 150 mM NaCl, 2.5% (v/v) glycerol, and 1 mM

CaCl2, pH 5.75. Protein purity was checked by silver-stainedSDS-PAGE.Effect of Calcium Chloride on Enzymatic Activities—See the

supplemental text.Standard Activity Determination—Standard activities were

determined as described previously (12) only in the presence ofsucrose.Acceptor Reactions with �N123-GBD-CD2—The reaction in

the presence of sucrose and maltose was performed usingstandard conditions for 24 h using 146 mM of maltose and 1.0unit/ml of enzymatic activity. Branching reactions of linear

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dextrans were carried out using standard conditions with 292mM sucrose and 2,470, 1,237, 463, 311, 123, 92, or 62 mM of70-kDa dextran acceptor (dextran concentrations expressed asanhydroglucosyl unit concentrations) with 1.5 units/ml of puri-fied enzyme. The reaction medium was analyzed by HPLC asdescribed by Brison et al. (12) to assay glucose, fructose, andleucrose production and sucrose depletion and by determining(i) glucose production rates (hydrolysis activity), (ii) fructoseproduction rates that reflect both �-(132) glucosylation andhydrolysis activity, and (iii) �-(132) linkage content (12).Approximately 10 mg of each purified branched dextran waspurified and analyzed by 1H NMR to determine its �-(132)linkage content (12).Steady-state Kinetics—The ��123-GBD-CD2 kinetic mech-

anism was investigated by steady-state kinetics following themethodology described by Brison et al. (12).Crystallization and Data Collection—Freshly purified

enzymewas concentrated using a centrifugal filter device (Ami-con Ultra, 4 Ultracel, 50 kDa; Millipore) to 3–4 mg/ml esti-mated by spectroscopy at 280nmwith theoreticalmolar extinc-tion coefficient and molecular weight calculated using theExPASy ProtParam tool. The protein to reservoir volume ratioin the 2-�l hanging-drop was 1:1. The crystals were obtainedfrom two crystallization conditions (I and II). In condition I,��123-GBD-CD2 enzyme crystallized either as needles or plateclusters over several weeks at 285 K using 17% (w/v) PEG 3350,0.2MNH4I, 80mMammoniumcitrate, 2% (v/v) glycerol, pH5.0,as precipitant. Streak seeding resulted in single or clusteredplate crystals with a thickness of 10–20�m.Crystals were cryo-protected in reservoir solution supplementedwith 15% glycerol(w/v) and then cryo-cooled in a gaseous nitrogen flux at 100 K.

The majority of the plate crystals usually diffracted to 5–8 Åresolution; a crystal (100� 100� 10�m3), obtained by co-crys-tallization with the glucohexaose �-D-Glcp-(136)-�-D-Glcp-(136)-�-D-Glcp-(136)-�-D-Glcp-(136)-�-D-Glcp-(134)-D-Glcp, diffracted anisotropically to 3.2 Å resolution in onedirection and to 3.4 Å in the other direction. This crystal wasused for data collection at the European Synchrotron RadiationFacility (Grenoble, France) at 100 K on Beam Line ID14-2. Thedatawere processed using iMOSFLMand SCALA (18–20). Ac-cording to the methodology for structure determination at lowresolution described by Brunger et al. (21), we fixed the resolu-tion limit at an I/�(I) cutoff of 1.6 (at 3.3 Å resolution).In condition II, pyramidal crystals grew over several weeks at

285 K in 15% PEG 3350 and 0.1 M NH4NO3. They were cryo-protected in 20% PEG 3350, 0.1 MNH4NO3, and 11% (v/v) glyc-erol. One of these crystals was used for data collection at theSoleil synchrotron (Gif-sur-Yvette, France) at 100 K on thePROXIMA1 beam line. The resulting 1.90 Å resolution data setwas indexed, integrated, and scaled using XDS (22).Structure Determination and Refinement—Details of data

collection, cell parameters and processing statistics are pre-sented inTable 1. The data set at 3.3Šresolutionwas the first tobe collected. CHAINSAW (23) was used to obtain a mixedhomologymodel for domains A, B, and C using the structure ofGTF180-�N (Protein Data Bank entry 3KLK) (8) and the��123-GBD-CD2 sequence. This model was used as a templatefor molecular replacement with PHASER (24). Four looselypacked molecules were found in the asymmetric unit. Modelbuilding and refinement were done using COOT (25) and REF-MAC5 (26), respectively, applying NCS restraints and resultedin an incomplete model.

TABLE 1Data collection and refinement statisticsThe numbers in parentheses refer to statistics for the outer resolution shell.

Triclinic crystals Orthorhombic crystals

Data collectionWavelength (Å) 0.933 0.954Cell dimensions, a, b, c (Å) and �, �, and � (°) 66.8, 140.0, 155.5, 85.4, 90.9, 76.9 68.2, 100.2, 187.2, 90.0, 90.0, 90.0Space group P1 P212121Molecules per asymmetric unit 4 1Resolution limit (Å) 51.6-3.3 (3.48-3.30) 46.0-1.9 (1.95-1.90)Reflections (total/unique) 153,233/80,648 610,291/98,989Completeness (%) 97.5 (97.0) 97.2 (97.0)Rmerge (%) 20.3 (53.3) 7.1 (45.4)cI/�(I) 4.1 (1.6)b 18.0 (4.4)Wilson B factor (Å2) 42.3 29.7

Refinement statisticsReflections (working/test) 76,146/4,029 94,038/4,950Rcryst/Rfree 0.224/0.291 0. 157/0.198Number of atoms 32,344 9,331Protein 8123/8106/8072/7952 8128Liganda 54 152Ion/water 4/33 2/1049B factor (Å2)Main chain 25.6 25.7Side chain 26.5 27.7Ion/water/ligand 30.0/20.0/30.0 30.6/34.6/54.7

Stereochemical quality of the modelRoot mean square deviation from bond lengths (Å) 0.009 0.020Root mean square deviation from angles (°) 1.107 1.835Ramachandran favored (%) 93.1 97.2Ramachandran allowed (%) 98.9 99.9Ramachandran disallowed (%) 1.1 0.1

a Glycerol or polyethylene glycol molecules.b Mean I/�(I).c Rmeas.

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The high resolution structure of ��123-GBD-CD2 wasobtained by molecular replacement using PHASER with theincomplete 3.3 Å resolution structure. Then ARP/wARP (27)was used to entirely rebuild the structure and place water mol-ecules. The final Rwork and Rfree values for the high resolutionstructure were 0.157 and 0.198, respectively. The high resolu-tion structure was in turn used to re-examine and complete thelow resolution structure leading to finalRwork andRfree values of0.224 and 0.291, respectively. Residue numbering refers to theprotein sequence of the full DSR-E dextransucrase sequenceavailable in UniProt (accession number Q8G9Q2). The coordi-nates and structure factors have been deposited in the ProteinData Bank (entries 3TTO and 3TTQ).Mutagenesis Studies—Mutants A2249W, G2250W, A2249D/

G2250W, and F2214N were constructed by inverse PCR using�N123-gbd-cd2 as template and the primers described in sup-plemental Table S2. E. coli TOP10 cells (Invitrogen) were usedas hosts for gene expression and mutant production. Themutants were tested in the presence of sucrose alone or with anadditional maltose acceptor or 1-kDa dextran acceptor. Thereaction products were analyzed by high performance anionexchange chromatography with pulsed ampero-metric detec-tion (HPAEC-PAD) and compared with those obtained withthe WT �N123-GBD-CD2 enzyme.Docking Study—Docking of isomaltotriose in a model of the

�N123-GBD-CD2 glucosyl enzyme intermediate is described inthe supplemental text.

RESULTS AND DISCUSSION

Construction and Production of Truncated GBD-CD2Enzyme—Attempts to crystallize the full-length GBD-CD2failed. We assumed that the length and the quite hydrophobicnature of GBD (849 amino acids) did not favor crystallization.Indeed, the N-terminal part of the protein contains 41 consec-utive repeat units rich in aromatic residues and homologous tothe cell wall binding units (Pfam family PF01473) ofC-LytA, theC-terminal choline-binding domain of Streptococcus pneu-moniae autolysin A (28, 29). Therefore, the C-LytA three-di-mensional structurewas used to find 12 truncation sites inGBD(supplemental Fig. S1). Of the 12 constructs, the shortest activetruncated form, �N123-GBD-CD2 (123 kDa; 1108 residues)with a glucan-binding domain reduced by 76%, was retained forfurther characterization. The purification procedure, whichincluded isolation of inclusion bodies, immobilized metal ionaffinity chromatography, and ion exchange chromatographyyielded 14 mg of pure �N123-GBD-CD2 enzyme/liter ofculture.Functional Characterization of �N123-GBD-CD2—Using

292 mM sucrose, �N123-GBD-CD2 mainly catalyzed sucrosehydrolysis. Several by-products, including leucrose (�-D-Glcp-(135)-D-fructopyranose), kojibiose (�-D-Glcp-(132)-D-Glcp),and traces of maltulose (�-D-Glcp-(134)-D-fructofuranose),were also obtained. They result from glucosyl transfer onto thefructose and glucose units released by hydrolysis (supplementalFig. S2A). In the presence of sucrose and maltose, the enzymeyielded the same products as with sucrose alone, indicating thatmaltose is not an acceptor of �N123-GBD-CD2 (supplementalFig. S2B).

�N123-GBD-CD2was also tested for its ability to branch dex-tran. 1H NMR spectra confirmed that �N123-GBD-CD2 cata-lyzes the formation of branched dextrans containing �-(132)glucosidic bonds (Fig. 1A). Branching amounts could be con-trolled by modulating the [sucrose]/[dextran] molar ratio (Fig.1B). As shown in Fig. 1C, when the dextran concentration wasincreased, glucose and leucrose production was reduced.Indeed, at low molar [sucrose]/[dextran] ratios (0.12 and 0.24),the glucosyl units from sucrose are almost exclusively trans-ferred onto dextran, similar to what has been observed forGBD-CD2 (11, 12).

�N123-GBD-CD2 displays Michaelis-Menten kinetics forsucrose hydrolysis. Km, SucH, and kcat,SucH values are 7.5 � 1.0mM and 76 s�1, respectively (Table 2). In the presence of dex-tran as acceptor, the initial velocities of the �-(132) glucosyla-tion obey a ping-pong bi-bimechanism. The catalytic constantskcat,T for �N123-GBD-CD2 and GDB-CD2 are not significantlydifferent. In contrast, the apparentKm,DexT andKm,SucT are 1.6-and 4.9-fold higher than those observed for GBD-CD2, respec-tively, indicating that the deleted part of the GBD may favorbinding of both sucrose and high molecular weight dextran.Deletion of the complete GBD (from Gln-1141 to Leu-1980)resulted in an almost inactive variant (11). The remaining partof GBD found in �N123-GBD-CD2 is thus necessary to main-tain enzymatic activity.Crystal Structures of �N123-GBD-CD2—The structure of

�N123-GBD-CD2 was solved at 1.90 Å resolution in its apoform. Refinement statistics are summarized in Table 1. Thepolypeptide chain (1043 residues) is organized in five distinctdomains named C, A, B, IV, and V (Fig. 2A), which are notconsecutively arranged along the peptide chain, similarly to thefold of other GH70 enzymes (Fig. 2B). A structure was alsosolved at 3.3 Å resolution in another space group. The super-imposition of the two structures of �N123-GBD-CD2 showedthat the positions of backbone atoms of domains IV and V areshifted by 1.8–7.0 Å (supplemental Fig. S3). In addition, noglucohexaosemolecule was identified in the enzyme active site,although the protein was co-crystallized with this substrate.At the interface of domains A and B, a heptacoordinated

metal ion is bound that is probably a calcium ion. Indeed, thebinding site is homologous to the Ca2�-binding site ofGTF180-�N in which Asp-2164 of �N123-GBD-CD2 replacesGlu-979 of GTF180-�N (8). Distances between ligands (i.e. car-bonyl group of Asp-2164, O�1 and O�2 of Asp-2170, carbonylgroup of Phe-2214, O�1 of Asn-2693, and twowatermolecules)and the metal ion range from 2.32 to 2.61 Å (supplemental Fig.S4). In addition, activitymeasurements showed that the activityof �N123-GBD-CD2 decreased by 14% in the presence of theCa2�-chelating agent EDTA. This is in agreement with calciumdependence (supplemental Table S3) (30).Individual Domain Description—Domain C consists of eight

anti-parallel� strands and includes amodifiedGreek keymotif.The function of this domain remains unknown.Domain A is the largest domain; it forms the catalytic core

together with elements from domain B. It comprises a (�/�)8barrel similar to that of GTF180-�N and GFT-SI glucansu-crases, which is circularly permuted compared with that ofGH13 enzymes (Fig. 3 and supplemental Fig. S5) (8, 9). Domain

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C is inserted between helix �8 and strand �1, and domain Bconnects strand �3 to helix �3. In domain A, many structuralfeatures distinguish �N123-GBD-CD2 from GTF180-�N andGTF-SI. First, helix �5, which is downstream from the putativecatalytic Glu-2248, is three residues shorter than that ofGTF180-�N and GTF-SI and adopts a different position (sup-plemental Fig. S5). Conversely, the loop from Gly-2731 to Ser-

2796 is 25 residues longer than the equivalent loop ofGTF180-�N (supplemental Fig. S5). Starting with two contig-uous�-helices (residuesGln-2734 toTyr-2739 andGln-2741 toLys-2750) followed by a �-hairpin covering helices �3, �4, and�5, it protrudes from domain B and contributes to domain A(Fig. 3). Loop Asp-2292 to Ile-2299, which connects helix �6 tostrand �7 of the (�/�)8 barrel, is also four residues longer than

FIGURE 1. A, anomeric region of 1H NMR spectra obtained at 298 K for purified �-(132) branched dextrans. Black, dextran 70-kDa standard; blue, dextran�-(132) branched at 11%; pink, dextran �-(132) branched at 19%; orange, dextran �-(132) branched at 33%; green, dextrans �-(132) branched between 35and 37%. B, percentage of �-(132) linkage as a function of [sucrose]/[dextran] ([Suc]/[Dex]) molar ratios used for the acceptor reactions. Empty and filled circlescorrespond to values obtained after 1H NMR and HPLC measurement, respectively, of the �-(132) linkage content in dextrans synthesized by �N123-GBD-CD2.Red crosses, 1H NMR results for dextrans branched by GBD-CD2 (12). C, effects of the [sucrose]/[dextran] molar ratio on �-(132) branched dextran yields. Thereactions were carried out at 292 mM sucrose and various dextran concentrations. Main final reaction products are residual sucrose, glucose (from sucrosehydrolysis), leucrose (from fructose glucosylation), and �-(132) branched dextran.

TABLE 2Comparison of the apparent kinetic parameters determined for sucrose hydrolysis (subscript H) and �-(132) dextran branching activities(subscript T) for GBD-CD2 and �N123-GBD-CD2

Apparent kinetic parameters GBD-CD2a �N123-GBD-CD2

In the presence of sucroseVmax H (�mol�min�1�mg�1 of purified enzyme) 34.6 � 0.5 36.3 � 0.6Km,SucH (mM) 10.8 � 0.8 7.5 � 1.0kcat,SucH (s�1) 109 76

In the presence of sucrose and dextran 70 kDaVmax T (�mol�min�1�mg�1 of purified enzyme) 303 � 5 462 � 45Km,SucT (mM) 42 � 2 206 � 34Km,DexT (mM of anhydroglucosyl units) 75 � 3 125 � 21Km,DexT (mM) 0.174 � 0.008 0.30 � 0.05kcat T (s�1) 970 947kcat T/Km,SucT (s�1�mM�1) 23 4.6kcat T/Km,DexT (s�1�mM�1) 13 7.6

a From Ref. 12.

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its equivalent in GTF180-�N (supplemental Fig. S5). LoopsGly-2731 to Ser-2796 and Asp-2292 to Ile-2299 are bothlocated at the upper part of the catalytic gorge, which is alsodelineated by a small subdomain inserted between strand �6and helix �7 (from Gly-2324 to Asn-2368). This small sub-domain comprises two helices, H1 and H2, also found in GTF-180-�N and GTF-SI (Fig. 3).Domain B is folded into a five-stranded �-sheet. A compari-

son with domain B of GTF180-�� shows that 94 C� of 99 C�superimpose well (root mean square deviation, 1.2 Å). How-ever, the loop inserted between Arg-2157 and Phe-2163, alsolocated at the upper part of the catalytic gorge, is 11 residuesshorter than the equivalent one of GTF180-�N (supplementalFigs. S5 and S6).Domain IV can be superimposed with that of GTF180-�N

with secondary structure elements conserved but slightlyshifted. A DALI analysis showed no significant structural sim-ilarities of this domain to other proteins in the Protein DataBank (31).With a global V shape, domain V adopts an original fold that

is similar only to domain V of GTF180-�N but not to any other

protein domain in the Protein Data Bank (DALI analysis) (Fig.4). Although in GTF180-�N both the N- and C-terminal partsof the peptide chain contribute to domain V, in �N123-GBD-CD2 it is only made up from residues of the N-terminal part. Itcomprises three subdomains exhibiting an organization inwhich a three-stranded �-sheet is connected to two consecu-tive �-hairpins. Each �-hairpin is constituted of two �-strandsof 3–6 residues separated by short loops of 2–11 amino acids.The two�-hairpins and the three-stranded�-sheet of each sub-domain are rotated by �120° with respect to each other.Sucrose Specificity at Subsites �1 and �1—The �N123-

GBD-CD2 active site forms a pocket into a large gorge(supplemental Fig. S7). Because ��123-GBD-CD2 andGTF180-�N both use sucrose as substrate, we superimposedthe structure of ��123-GBD-CD2 with that of the GTF180-�N-sucrose complex (Protein Data Bank entry 3HZ3) toinvestigate the functional role of specific residues lining theactive site of ��123-GBD-CD2. As shown in Fig. 5, 17 resi-dues defining subsites �1 and �1 (32) in �N123-GBD-CD2align well with the corresponding residues of GTF180-�N(root mean square deviation, 0.53 Šon C�). At subsite �1,

FIGURE 2. A, stereo view of �N123-GBD-CD2 domain organization. Magenta, domain C; blue, domain A, which includes the (�/�)8 barrel; green, domain B; yellow,domain IV; red, domain V. B, schematic representation of the domain arrangement along the polypeptide chains of crystallized GH70 glucansucrases, from leftto right, GTF180-�N, GTF-SI, and �N123-GBD-CD2. The color code is identical to that for A. Striped red, parts of domains V of GTF-SI and �N123-GBD-CD2, whichare not visible in the electron density map; white, purification tag.

FIGURE 3. Stereo view of the secondary structure elements of �N123-GBD-CD2 domains A and B. For domain A, blue shows the (�/�)8 barrel, cyan showsthe subdomain H1-H2, and purple shows the loop Gly-2731 to Ser-2796 protruding from domain B and contributing to domain A. Domain B is green.

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10 conserved residues (Arg-2208, Asp-2210, Glu-2248, His-2321, Asp-2322, Asn-2596, Asp-2643, Tyr-2650, Asp-2689,and Gln-2694) correspond to residues that interact withthe glucosyl moiety of sucrose in GTF180-�N (Fig. 5). In

particular, residues Asp-2210 and Glu-2248 coincide withthe nucleophile and the general acid/base catalyst ofGTF180-�N and GH13 enzymes (8, 33, 34) and are in a posi-tion to play the same role in ��123-GBD-CD2. Likewise,

FIGURE 4. Upper panel, stereo view of the secondary structure elements of domain V (truncated glucan-binding domain) of �N123-GBD-CD2. The three-stranded �-sheets and �-hairpins of the three subdomains are represented in red and salmon, respectively. Lower panel, sequence alignment of the domain V.The underlined and colored residues are �-strands; N-terminal residues in italics are not visible in the electron density map.

FIGURE 5. Stereo view of subsites �1 and �1 of �N123-GBD-CD2, with sucrose from the GTF180-�N-sucrose complex superimposed. The catalyticresidues are Asp-2210 (nucleophile), Glu-2248 (acid/base), and Asp-2322 (transition state stabilizer). Sucrose is shown with yellow carbons. Residues of theinactive GTF180-�N mutant (D1025N) that interact with sucrose (8) are represented in gray. The carbon atoms of their structural equivalents in �N123-GBD-CD2are shown in blue (domain A), cyan (subdomain H1-H2), and green (domain B).

TABLE 3Characterization of single and double mutants targeting nonconserved residues of subsite �1

Relative activitya Sucrose Acceptor concentration Hydrolysis yieldbSucrose isomer

yieldcTransfer ontoacceptord

% mM mM % % %�N123-GBD-CD2WT 100 146 87 13

146 1-kDa dextran, 146 20 1 79146 Maltose, 146 88 12 NDe

�N123-GBD-CD2 A2249W 21.7 146 89 11146 1-kDa dextran, 146 24 2 74146 Maltose, 146 90 10 ND

�N123-GBD-CD2 G2250W 1.1 146 89 11146 1kDa dextran, 146 45 4 51146 Maltose, 146 90 10 ND

�N123-GBD-CD2 A2249D-G2250W 89.3 146 90 10146 1-kDa dextran, 146 36 4 60146 Maltose, 146 90 10 ND

�N123-GBD-CD2 F2214N 18.6 146 100 ND146 1-kDa dextran, 146 100 ND ND146 Maltose, 146 100 ND ND

a The relative activity was determined as the ratio of sucrose consumption (initial rate) of each mutant versus sucrose consumption (initial rate) of �N123-GBD-CD2.b Hydrolysis yield � Glc (mol)/sucrose consumed (mol) �100.c Sucrose isomer � Glc incorporated into leucrose (mol)/sucrose consumed (mol) � 100.d Transfer onto acceptor � 100 � hydrolysis ratio % � sucrose isomer yield %.e ND, not detectable.

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Asp-2322 is equivalent to the transition state stabilizing res-idue Asp-1136 of GTF180-�N.At subsite �1, seven residues of GTF180-�N have interac-

tionswith the fructosyl ring of sucrose (8). Three of them (Lue-2166, Leu-2167, and Gln-2326) are conserved between��123-GBD-CD2 and GTF180-�N. The two leucine resi-dues are involved in van der Waals interactions with thefructosyl moiety of sucrose. Residue Gln-2326 is well posi-tioned to make a hydrogen bond with the C6 hydroxyl of thefructosyl moiety. In contrast, two residues of GTF180-�Nsubsite �1 (Asn-1029 and Trp-1065), which are H-bonded

to the fructosyl ring of sucrose, have no equivalent in �N123-GBD-CD2. It is noteworthy that these residues are usuallyconserved in the GH70 family except for GBD-CD2. Asshown in Fig. 5, Asn01029 is substituted by Phe-02214, andthe position corresponding to Trp-1065 is occupied by Ala-2249 and Gly-2250. None of these residues can makeH-bonds with the fructosyl residue. Only Lys-2323 mayestablish a hydrogen bond with the O4 of the fructosyl unit,suggesting a weaker sucrose binding in �N123-GBD-CD2.This may explain why �N123-GBD-CD2 has a 32-fold higherapparent Km for sucrose than GTF180-�N (6).Acceptor Recognition—In GH70 enzymes, subsite �1 not

only has to accommodate the fructosyl unit of the donor sub-strate but must also bind acceptors. We have shown that�N123-GBD-CD2 does not glucosylate maltose. This disaccha-ride is known to be the most efficient acceptor for glucansu-crases (35). Analysis of the GTF180-�-maltose complex struc-ture (Protein Data Bank entry 3KLL) showed that theinteraction between themaltose residue andTrp-1065 is essen-tial for maltose glucosylation, which results in the formation ofpanose (�-D-Glcp-(136)-�-D-Glcp-(134)-D-Glcp) (8). Trp-1065 is not conserved in �N123-GBD-CD2, and there is nostacking platform at this position to keep the nonreducing endresidue of maltose in a correct position for glucosylation.Mutagenesis Studies—To further investigate the possible

involvement in acceptor binding of the nonconserved residuesAla-2249, Gly-2250, and Phe-2214, we constructed mutantsA2249W, G2250W, A2249D/G2250W, and F2214N, in whichthe amino acids were replaced by the corresponding residues ofGTF180-�N. Because the structural alignment revealed thatTrp-1065 ofGTF180-�Noccupies a position in betweenAla-2249 andGly-2250, bothAla-2249 andGly-2250were individually replacedby a tryptophan residue. Themutants were tested in the presenceof sucrose alone or with additional acceptors (maltose or 1-kDadextran). When using only sucrose as a substrate, all of themutantswereunable to formpolymerandshowedreducedhydro-lytic activity (Table 3). Apparently, subsite�1 is tolerant tomuta-tions with regard to sucrose utilization.

FIGURE 6. Isomaltotriose docking in the catalytic groove of the modeledglucosyl-enzyme intermediate of �N123-GBD-CD2. Two binding modeswere found that allow glucosylation through �-(132) linkage formation ontothe central glucosyl unit (A) or the nonreducing end extremity (B). The cova-lently linked glucosyl unit in subsite �1 is represented in gray, and isomalto-triose is in yellow. The distances between O2 atom of isomaltotriose and C1atom of the glucosyl enzyme intermediate (in Å) are shown in yellow. Domaincoloring is as in Fig. 3.

FIGURE 7. Stereo view of the catalytic gorges of GTF180-�N and �N123-GBD-CD2. For clarity, only different residues or residues adopting differentconformations are shown. Backbone atoms of �N123-GBD-CD2 are depicted in light blue. Residues from domain A of �N123-GBD-CD2 are depicted in blue, cyan,and purple (see Fig. 3). Residues from domain B of �N123-GBD-CD2 are shown in green. Gray residues belong to GTF180-�N. Sucrose from GTF180-�N-sucrosecomplex (Protein Data Bank entry 3HZ3) in subsites �1 and �1 is represented as yellow carbons.

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Withmaltose, none of themutants showed transferase activ-ity. In particular, the introduction of a tryptophan residue atposition 2249 and/or 2250 did not promote the transfer reac-tion. Likely, other changes at the acceptor binding subsite �1are necessary to generate such activity. However, when 1-kDadextran was added as an acceptor, mutants at positions 2249and/or 2250were still able to catalyze the formation of�-(132)branches like the WT enzyme (supplemental Fig. S8). Appar-ently these positions are not directly involved in regiospecific-ity. In contrast, mutant F2214N was unable to use 1-kDa dex-tran as an acceptor (Table 3 and supplemental Fig. S9). Thus,this residue is critical for dextran binding and branching.To catalyze glucosyl transfer onto dextrans, �N123-GBD-

CD2must first bind sucrose and then catalyze the formation ofthe covalent �-D-glucosyl enzyme complex. Fructose must bereleased from subsite �1 to allow the acceptor to enter. Thedextran glucosyl residue that will be branched must bind insubsite�1with its C2 hydroxyl group properly oriented towardtheC1 of the�-D-glucosyl-enzyme intermediate. In accordancewith the ping-pong bi-bi mechanism of �N123-GBD-CD2, theproduct has to leave the active site after branching to allow thebinding of a new sucrose molecule. How acceptor moleculesare accommodated in the catalytic gorge in a position to be�-(132) glucosylated remains to be elucidated. Despitenumerous attempts to crystallize complexes of �N123-GBD-CD2 with isomaltotriose, linear, or �-(132) branched gluco-oligosaccharides, we were not successful. Docking of isomalto-triose was thus attempted in a model of the glucosyl-enzymeintermediate of ��123-GBD-CD2. Twomain groups of dockedisomaltotriose were obtained with a glucosyl unit of the accep-tor chain appropriately oriented to allow the formation of an�-(132) linkage. In the first group, the central glucosyl unit ofisomaltotriose is in a position to be �-(132) glucosylated (Fig.6A), with a shortest distance of 4.10 Šbetween the O2 atom ofthe acceptor glucosyl unit and the C1 atom of the glucosylenzyme intermediate. In the second group, the glucosyl unit atthe nonreducing extremity would be glucosylated with a dis-tance between O2 and C1 of 3.45 Š(Fig. 6B). These modelsreveal that �-(132) branching occurring at either the nonreduc-ing end or at a residue of the dextran backbone is feasible. More-over, structural analysis of �N123-GBD-CD2 has identified resi-dues that line its catalytic gorge and that could be involved indextranbindingorenzymeregiospecificity (Fig. 7). Indeed, one ofthese residues, Phe-2214, was shown by site-directedmutagenesis to be crucial for dextran accommodation. Theroles of the other residues, as well as residues in the threeloops specific for GBD-CD2, will now be explored to furtherdeepen our understanding of the mechanism and specificityof the enzyme.

Acknowledgments—We thank the staff of Beam Line ID14-2 at theESRF (Grenoble, France) and Pierre Legrand from the PROXIMA1beam line at the SOLEIL synchrotron (Gif-sur-Yvette, France) fordata collection facilities and assistance.We greatly thank NellyMon-ties, Pierre Escalier, Sandra Pizzut-Serin, and Virginie Rivière fortechnical assistance.

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